Impact of Contaminating DNA in Whole-Genome Amplification Kits Used for Metagenomic Shotgun Sequencing for Infection Diagnosis

Abstract

Whole-genome amplification (WGA) is a useful tool for amplification of very small quantities of DNA for many uses, including metagenomic shotgun sequencing for infection diagnosis. Depending on the application, background DNA from WGA kits can be problematic. Three WGA kits were tested for their utility in a metagenomics approach to identify the pathogens in sonicate fluid comprised of biofilms and other materials dislodged from the surfaces of explanted prosthetic joints using sonication. The Illustra V2 Genomiphi, Illustra single cell Genomiphi, and Qiagen REPLI-g single cell kits were used to test identical sonicate fluid samples. Variations in the number of background reads, the genera identified in the background, and the number of reads from known pathogens known to be present in the samples were observed between kits. These results were then compared to those obtained with a library preparation without prior WGA using an NEBNext Ultra II paired-end kit, which requires a very small amount of input DNA. This approach also resulted in the presence of contaminant bacterial DNA and yielded fewer reads from the known pathogens. These findings highlight the impact that WGA kit selection can have on metagenomic analysis of low-biomass samples and the importance of the careful selection and consideration of the implications of using these tools.

Keywords: metagenomics; prosthetic joint infection; whole-genome amplification.

FIG 1

Beta diversity of samples by sample source versus WGA kit. The beta diversity of the gene content of the samples was determined using QIIME and plotted on a principal coordinate analysis plot. Samples are colored according to the WGA kit used for amplification (A) or by template sample source (B). NT, no template.